The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
Carbon fixation is commonly performed by autotrophic plants and microorganisms such as chemoautotrophic and phototrophic microorganisms by incorporation of CO2 into more complex molecules, typically via the CBB cycle (Calvin-Benson-Bassham cycle, also known as reductive pentose phosphate cycle). In this cycle, three molecules of CO2 are fed through a series of enzymatic reactions that generate a variety of phosphorylated compounds, which when coupled to glycolysis, produce a single molecule of acetyl-CoA.
Due in part to this ability to fix atmospheric carbon, a number of methods have been developed to divert the products of these reactions into commercially valuable materials such as alcohols, fuels, and biodegradable plastics. For example, WO 2009/098089 to Duhring et al. teaches certain genetic modifications of photosynthetic autotrophs to enhance activity or to modify cofactor specificity of enzymes involved in the production of specific metabolites, and to overexpress enzymes involved in ethanol synthesis from such metabolites. Similarly, US 2012/0142066 to Baier et al. teaches genetically engineered photoautotrophs to enhance ethanol production by overexpression of enzymes involved in the ethanol synthesis and reduction of activity of enzymes that utilize intermediates in the ethanol synthesis in alternate pathways. While at least somewhat effective, such methods are typically limited to ethanol production and, in addition, fail to address issues of inefficient fixation of CO2.
More recently, an improved CO2 fixation process was reported where a non-oxidative glycolysis (NOG) step used three molecules of fructose-6-phosphate to produce three molecules of acetyl-CoA, and where further carbon rearrangement reactions were needed to regenerate two molecules of fructose-6-phosphate (see e.g., Nature 502, 693-697 (31 Oct. 2013)). Thus, one molecule of ‘surplus’ fructose-6-phosphate from the CBB cycle was used to form three molecules of acetyl-CoA, and six molecules of CO2 were needed to obtain via glyceraldehyde-3-phosphate the fructose-6-phosphate. Therefore, viewed from a different perspective, the NOG pathway required a transaldolase key step (C6+C4→C3+C7) and subsequent conversion of glyceraldehyde-3-phosphate to fructose-6-phosphate before the fructose-6-phosphate enters the non-oxidative glycolysis. However, while at least somewhat improving CO2 fixation, additional genetic modifications were required to absorb and reconfigure the erythrose-4-phosphate byproduct from the acetyl-CoA generation and to ultimately regenerate fructose-6-phosphate.
Regardless of the efficiency of CO2 fixation, at least some of the microorganisms that produce value products (e.g., alcohols, polyhydroxyalkanoates, etc.) often require substantial quantities of nitrogen (typically in form of ammonia) to grow to a desirable cell density. Unfortunately, relatively high nitrogen levels favor cell growth over value production, and the cells are typically shifted to nitrogen-limiting or nitrogen depletion conditions to shift the cells to value product formation. However, low nitrogen levels in the growth medium have also been found to reduce the rate of CO2 fixation, likely by feedback inhibition of a CBB cycle metabolite, limiting overall yield of the value products.
Thus, there is still a need for methods and compositions that permit efficient carbon fixation by autotrophic organisms under conditions that also permit efficient production of value added materials without imposing undue metabolic burden and additional catalytic activities onto a cell. Moreover, there is also a need to provide metabolically engineered cells that can produce value products at a high rate under nitrogen-limiting or nitrogen depletion conditions without feedback inhibition by a CBB cycle metabolite that accumulates under such conditions.